The present disclosure relates generally to the field of DNA analysis. More particularly, the present disclosure relates to methods, preservative reagents and systems for preserving DNA in bodily fluids for subsequent testing and analysis.
Modern testing and treatment procedures have successfully reduced the prevalence and severity of many infectious diseases. For example, sexually-transmitted disease (STD) clinics regularly screen and treat patients for such diseases as gonorrhea and Syphilis. It is now well-known to identify infectious agents such as gonococci by analyzing a DNA sample. A genetic transformation test (GTT), such as Gonostat(copyright) (Sierra Diagnostics, Inc., Sonora, Calif.), can be used to detect gonococcal DNA in specimens taken from the urethra of men, and the cervix and anus of women, according to Jaffe H W, Kraus S J, Edwards T A, Zubrzycki L. Diagnosis of gonorrhea using a genetic transformation test on mailed clinical specimens, J Inf Dis 1982; 146:275-279. A similar finding was also published in Whittington W L, Miller M, Lewis J, Parker J, Biddle J, Kraus S. Evaluation of the genetic transformation test,. Abstr Ann Meeting Am Soc Microbiol 1983; p. 315.
The GTT is a test for biologically active or native DNA. For example, the Gonostat(3) GTT can be used to detect DNA such as gonococcal DNA in urine specimens. The Gonostat(copyright) assay uses a test strain, N. Gonorrhoeae, ATCC 31953. This test strain is a mutant that is unable to grow into visible colonies on chocolate agar at 37xc2x0 C. in 5% CO2. Gonococcal DNA extracted from clinical material can restore colony growth ability to this test strain. The Gonostat(copyright) assay is discussed in Zubrzycki L, Weinberger S S, Laboratory diagnosis of gonorrhea by a simple transformation test with a temperature-sensitive mutant of Neisseria gonorrhoeae. Sex Transm Dis 1980; 7:183-187.
It is not always possible to immediately test a patient for the presence of such an infectious agent. For example, clinical laboratories are not readily found in many rural or underdeveloped areas. In such circumstances, it is necessary to transport patient test specimens to a laboratory for analysis. It is therefore desirable to preserve such specimens for subsequent analysis with a GTT or other testing procedure.
Urine specimens are frequently practical and convenient for use in diagnoses of an infection, such as gonorrhea A urine specimen can be collected by a patient, therefore avoiding the invasion of privacy and discomfort accompanying collection of other specimens, such as blood specimens, urethral cultures, or cervical cultures. Collection of a urine specimen by the patient also reduces the work load of the staff in the clinic or office.
DNA culture results of urine from males are quite sensitive when the urine is cultured within two hours of collection. Such results can approach 92% to 94%, or even 100%, as described in Schachter J. Urine as a specimen for diagnosis of sexually transmitted diseases. Am J Med 1983; 75:93-97. However, the culture results of urine from females are not very reliable, even when cultured within two hours. According to Schachter, only 47% to 73% of female urine cultures are positive relative to the culture results of cervical and anal specimens. Furthermore, it is known that culture results from any anatomic site are not 100% sensitive. (See, for example, Johnson D W, Holmes K K, Kvale P A, Halverson C W, Hirsch W P. An evaluation of gonorrhea casefinding in the chronically infected male. Am J Epidemiol 1969; 90:438-448; Schmale J D, Martin J E, Domescik G. Observations on the culture diagnosis of gonorrhea in women. JAMA 1969; 210:213-314; Caldwell J G, Price E V, Pazin G J, Cornelius E C. Sensitivity and reproducibility of Thayer-Martin culture medium in diagnosing gonorrhea in women. Am J Gynecol 1971; 109:463-468; Kieth L, Moss W, Berger G S. Gonorrhea detection in a family planning clinic: A cost-benefit analysis of 2,000 triplicate cultures. Am J Obstet Gynecol 1975; 121 :399-403; Luciano A A, Grubin L. Gonorrhea screening. JAMA 1980; 243:680-681; Goh B T, Varia K B, Ayliffe P F, Lim F K. Diagnosis of gonorrhea by gram-stained smears and cultures in men and women: Role of the urethral smear. Sex Trans Dis 1985; 12:135-139.
Currently, urine specimens must be tested quickly for the presence of naked gonococcal DNA. Naked DNA is intact double stranded DNA which is released from viable gonococci. Such naked DNA can be found in the urine of an infected patient. However, enzymes in urine rapidly destroy any DNA present in the specimen. The DNA is either denatured, broken into single strands or totally destroyed by the enzymatic activity. This destruction of the DNA can effectively inactivate the naked gonococcal DNA for purposes of testing.
In a test such as the GTT, inactivation beyond the limits of detection is determined by the inherent genetic needs for select gene sequences of the Gonostat mutant strain used in the Gonostat test. For example, the Gonostat transformation assay is a very sensitive measurement tool for nucleic acid protection. In the GTT, the Gonostat organism must have approximately 1 picogram of native DNA to transform. This amount is equal to the presence of approximately 30 gonorrhea bacteria in an inoculum. The average clinical infection has 103-105 such organisms.
The destruction of DNA by enzyme activity in a urine specimen increases with time. For example, naked gonococcal DNA in a urine specimen that is stored in excess of two hours is inactivated beyond the limits of detection of the GTT. As a result, the testing of urine specimens for DNA is very time-sensitive. For example, DNA-based tests such as the polymerase chain reaction (PCR), the ligase chain technology (LCx) test of Abbott Laboratories, Abbott Park, Ill., and the GTT all must be performed on a urine specimen within approximately two hours. FIG. 1 is a graph of DNA concentration in unpreserved urine according to the prior art, demonstrating DNA destruction over time. The gonococcal DNA concentrations of ten different types of urine specimens were tested using a GTT at hourly intervals, commencing one hour from time of inoculation. Approximately 200 transformants were counted at the one hour measurement. However, for all specimens, the number of transformants declined by more than 100% within one hour of this initial measurement. The number of transformants approached zero within the two hours of the initial measurement, FIG. 2 is a graph of eight day serial data on unpreserved urine according to the prior art, further illustrating DNA destruction in unpreserved samples. Approximately seven transformants were counted at the one day measurement. However, by the second day, testing indicated that the biologically active DNA in the unpreserved urine had been totally destroyed by enzyme activity.
Tests such as the GTT can also be used to detect DNA in such bodily fluids and excretions as blood, blood serum, amniotic fluid, spinal fluid, conjunctival fluid, salivary fluid, vaginal fluid, stool, seminal fluid, and sweat. FIG. 3 is a graph of DNA concentration in unpreserved serum according to the prior art, demonstrating DNA destruction over time. The gonococcal DNA concentrations of normal and abnormal serum of both male and female were tested at hourly intervals, commencing from the time of inoculation. Approximately 100 transformants were counted at the one hour measurement. However, for all specimens, the number of transformants declined by more than 100% within three hours of this initial measurement. The number of transformants approached zero within the eight hours of the initial measurement.
Another test that can be used to identify DNA in a bodily fluid specimen is the PCR test. PCR testing uses discrete nucleic acid sequences and therefore can be effective even in the absence of intact DNA. FIG. 4 is a graph of PCR detection of MOMP Chlamydia in unpreserved urine according to the prior art, demonstrating DNA destruction over time. In PCR testing of an unpreserved urine specimen, four PCR absorbances were observed one hour after the addition of the MOMP Chlamydia. However, the number of PCR absorbances declined 100%, to two, when tested at two hours, and to zero by the third hour. This testing indicates that, even though PCR testing doesn""t require intact DNA, the enzymatic activity of urine rapidly destroys even discrete nucleic acid sequences 45 within approximately three hours.
Unfortunately, practical and effective techniques for preserving DNA in certain bodily fluids have not been readily available. For example, one method used to deactivate urine enzymes is heating. In an experiment, urine was heated for five minutes in a boiling water bath (100xc2x0 C.) and then cooled. Naked DNA and DNA released from gonococcal cells that were subsequently added to this urine were not deactivated. This suggests that the deoxyribonuclease component in urine is a protein(s).
However, heating can denature DNA that is already present in the urine specimen, including gonococcal DNA, as well as the DNA of Haemophilus influenzoe and Bacillus subtilis. Thus, heating is not an appropriate method for preserving a patient urine specimen to test for the presence of such DNA.
In other known DNA assay systems, it is known to add detergents or other chemicals to assist in the detection of DNA. For example, in the DNA assay system described in Virtanen M, Syvanen A C, Oram J, Sodurlund H, Ranki M. Cytomegalovirus in urine: Detection of viral DNA by sandwich hybridization. J Clin Microbiol. 1984; 20:1083-1088, sarkosyl was used to detect cytomegalovirus (CMV) in urine by hybridization. In Boom R, Sol C J A, Salimans M M M, Jansen C L, Wertheim-van Dillen P M E, van der Noordaa J. Rapid and simple method for purification of nucleic acids. J Clin Microbiol 1990; 28:495-503, guanidinium chloride in urine was used to purify nucleic acids as assayed by gel electrophoresis. Although the reason for their use in these studies was not stated, the chemicals inactivated the deoxyribonuclease activity in urine that would have interfered with those assay systems.
It would therefore be advantageous to provide a method and system for preserving DNA in a bodily fluid such as urine, blood, blood serum, amniotic fluid, spinal fluid, conjunctival fluid, salivary fluid, vaginal fluid, stool, seminal fluid, and sweat, such that the efficacy of the DNA assays, e.g., the PCR, LCx, and the GTT is optimized.
The present disclosure relates to methods, systems and reagents for preserving nucleic acids, e.g., DNA and RNA, in bodily fluids. In one advantageous embodiment, the invention is directed to the preservation of nucleic acids in urine. In another advantageous embodiment, the invention enables the molecular assay of nucleic acids in other bodily fluids and excretions, such as blood, blood serum, amniotic fluid, spinal fluid, conjunctival fluid, salivary fluid, vaginal fluid, stool, seminal fluid, and sweat to be carried out with greater sensitivity, as the methods and preservatives of the invention have been found to surprisingly increase the signal obtained with such nucleic acid testing methods as polymerase chain reaction (PCR), LCx, and genetic transformation testing (GTT). An unexpected advantage of the invention is that hybridization in such nucleic acid testing methods is improved compared to when such nucleic acid testing methods are carried out without employing the present invention.
In an embodiment, the invention relates to methods of preserving a nucleic acid in a fluid such as a bodily fluid, including providing a nucleic acid preservative solution comprising an amount of a divalent metal chelator selected from ethylenediaminetetraacetic acid (EDTA), [ethylenebis(oxyethylenenitrilo)]tetraacetic acid (EGTA) and 1,2-bis(2-aminophenoxy)ethane-N,N,Nxe2x80x2,Nxe2x80x2-tetraacetic acid (BAPTA), or salts thereof; and an amount of at least one chelator enhancing component selected from lithium chloride, guanidine, sodium salicylate, sodium perchlorate, and sodium thiocyanate; and adding the nucleic acid preservative to the fluid, e.g., a bodily fluid. The amount of the divalent metal chelator is generally in the range of from about 0.001M to 0.1M, and the amount of the chelator enhancing component is generally in the range of from about 0.1M to 2M. The amount of chelator enhancing component is more desirably at least 1M in the preservative solution, and the divalent metal chelator is desirably present in an amount of at least about 0.01M.
In another embodiment, the invention relates to nucleic acid preservative solutions comprising an amount of a divalent metal chelator selected from EDTA, EGTA and BAPTA, and salts thereof, and an amount of at least one chelator enhancing component selected from lithium chloride, guanidine, sodium salicylate, sodium perchlorate, and sodium thiocyanate. The amount of the divalent metal chelator is generally in the range of from about 0.001M to 0.1M, and the amount of the chelator enhancing component is generally in the range of from about 0.1M to 2M. The amount of chelator enhancing component is more desirably at least 1M in the preservative solution, and the divalent metal chelator is desirably present in an amount of at least about 0.01M.
The methods and preservatives of the invention may further include an amount of at least one enzyme inactivating component such as manganese chloride, sarkosyl, or sodium dodecyl sulfate, generally in the range of about 0-5% molar concentration.
In yet another aspect the invention relates to a method of improving the signal response of a molecular assay of a test sample, including providing a nucleic acid preservative solution comprising an amount of a divalent metal chelator selected from EDTA, EGTA and BAPTA, and salts thereof; and an amount of at least one chelator enhancing component selected from lithium chloride, guanidine, sodium salicylate, sodium perchlorate, and sodium thiocyanate; adding the nucleic acid preservative to a test sample to provide a preserved test sample; extracting molecular analytes of interest, e.g., DNA, from the preserved test sample, and conducting a molecular assay on the extracted molecular analytes of interest. The amount of the, divalent metal chelator is generally in the range of from about 0.001M to 0.1M, and the amount of the chelator enhancing component is generally in the range of from about 0.1M to 2M. The chelator enhancing component is more advantageously one or more of sodium perchlorate, sodium thiocyanate, guanidine, and lithium chloride. The amount of chelator enhancing component is more desirably at least 1M in the preservative solution, and the divalent metal chelator is desirably present in an amount of at least about 0.01M. Signal response is believed to be enhanced in part due to enhanced hybridization as a result of the use of the reagents of the present invention.
Use of the methods and preservatives disclosed herein eliminate enzymatic destruction of the nucleic acid of interest in the bodily fluid. The preservative can optionally be provided in solid or gaseous forms. While the methods and preservatives of the invention are useful in preserving all types of nucleic acids, e.g., RNA and DNA, including human DNA, and bacterial, fungal, and viral DNA, the invention is especially advantageous for use in preserving prokaryotic DNA, e.g., gonococcal DNA, DNA of Haemophilus influenzoe and Bacillus subtilis. Nucleic acids in a bodily fluid are preserved for testing for a significantly longer period of time than that permitted by the prior art. While the maximum time between collecting, mailing, and testing patient specimens is expected to be approximately six days, the invention is effective beyond that period of time.
A further aspect of the invention relates to methods of improving hybridization of nucleic acids, including contacting a test nucleic acid with a nucleic acid preservative solution comprising an amount of a divalent metal chelator selected from ethylenediaminetetraacetic acid (EDTA), ethylenebis(oxyethylenenitrilo)]tetraacetic acid (EGTA) and 1,2-bis(2-aminophenoxy)ethane-N,N,Nxe2x80x2,Nxe2x80x2-tetraacetic acid (BAPTA), or salts thereof in the range of from about 0.001M to 0.1M; and an amount of at least one chelator enhancing component selected from lithium chloride, guanidine, sodium salicylate, sodium perchlorate, and sodium thiocyanate in the range of from about 0.1M to 2M, such that a test solution is formed; and contacting the test solution with a target nucleic acid under conditions favorable for hybridization, such that hybridization occurs.